Using featureCounts on HISTA2 aligned data of the tomato genome

[Antzel]

Hello there.

I am new with Bioinformatics and I am currently working on quantifying my genes in the RNA data that I received.
My workflow is as follows:
-Illumina sequenced data was received
-FastQC was done to determine quality
-Trimmomatic was used to trim my data
-HISAT2 was used to allign my data to a genome, paired ended data
Now I am at the featurecounts step, where I am currently getting 0% alignment.

Here is some information on what I have done so far:

$ featureCounts -p -O -T 4 --countReadPairs -a /home/antzeltheron/RNAseq_pipeline/HISAT2/ITAG4.0_gene_models.gtf -o /home/antzeltheron/RNAseq_pipeline/quants/ResC58_15_Rep1_R1_featurecounts.txt /home/antzeltheron/RNAseq_pipeline/HISAT2/ResC58_15_Rep1.sam

This gives me the following results:



As you can see, I have no successful alignments

Here are the HISAT2 results of 2 samples I ran:




For the HISAT run, the genome data I used was ITAG4.0_cDNA.fasta, which I used to build my index (hisat2_build command) which then created 8 files of .ht2 format.

Here is the SAM file after the HISAT2 run (only a few first lines):



And the GTF file's first 20 lines (ITAG4.0_gene-models.gtf):

I hope this is enough information.
I am really struggling to figure out how to get successful alignment.
Assistance will be greatly appreciated.

[crazyhottommy]

Hi,
can you please check the chromosome names in the Sam file and the gtf files are concordant?

The gtf file uses SL4.00ch00 etc. I am not familiar with plant genome, but that could be the reason.

Hope it helps.

Tommy